Symbiosis of E. coli and the Intestinal Microbiota in a Mouse Model

描述（由申请人提供）：人类肠道的微生物群落组成受到广泛关注，现在已经得到了相当好的表征。然而，单个微生物在肠道生态系统中的功能作用及其群落成员的动态相互作用在很大程度上仍然没有被表征，包括那些驱动资源竞争的相互作用（即，食物网）和那些为社区的成功创造有利条件的（例如，厌氧）。解决这些问题的实验不能在人类身上进行;它们需要一种动物模型，可以测试支撑人类微生物组的基本生态学原理。十多年来，我们一直使用链霉素处理的小鼠肠道定植模型来表征大肠杆菌在肠道中的功能作用。我们发现不同的E.大肠杆菌菌株执行不同的营养程序，使它们能够共同定植在肠道中。重要的是，我们了解到，E。大肠杆菌呼吸氧气，从而降低盲肠的氧张力，创造有利于主要厌氧微生物群落生长的条件。因此，我们假设E.大肠杆菌与厌氧菌共生。由于厌氧菌对氧敏感，我们预测肠道的群落组成至少部分取决于E.杆菌自E.大肠杆菌不能水解复杂的多糖，而多糖是肠道中的主要营养来源，大肠杆菌只能在降解产物上生长，我们预测厌氧多糖降解释放单糖，交叉喂养大肠杆菌。杆菌而且，由于不同的E.大肠杆菌菌株在肠道中执行不同的营养程序，我们预测每个大肠杆菌菌株在肠道中执行不同的营养程序。大肠杆菌菌株将与微生物群落的不同亚群相互作用。在这里，我们概述了一个研究计划，旨在测试这些预测。在目标1中，我们将使用16 S rRNA基因标签的高通量测序和分子系统发育分析来检查链霉素处理的小鼠肠道的微生物组成，因为它受到E.大肠杆菌定殖。利用等基因E.大肠杆菌菌株，可以和不能呼吸氧气，我们将测试的预测，氧气清除在肠道影响组成的厌氧微生物群落。此外，我们还将检验不同大肠杆菌菌株在肠道定植的预测。大肠杆菌在肠道中消耗不同的营养物质，会影响微生物群落的组成。在目标2中，我们将测试不同E.大肠杆菌菌株与微生物群落的不同成员物理关联，通过使用激光捕获显微切割从肠道取样的微生物的16 S分析。此外，我们将描述营养流之间的E。大肠杆菌和肠道微生物群落的单个成员共培养。目的3：确定大肠杆菌的表面结构，包括O-多糖、鞭毛和荚膜，是否能靶向大肠杆菌。大肠杆菌到特定的微生境，在那里它们可以与微生物群的不同成员相互作用。因此，我们将描述E.大肠杆菌与肠道中的微生物群落。

DESCRIPTION (provided by applicant): The microbial community composition of the human intestine is receiving a lot of attention and now is fairly well characterized. Yet the functional roles of individual microorganisms in the intestinal ecosystem and the dynamic interactions of its community members remain largely uncharacterized, including those interactions that drive competition for resources (i.e., the food web) and those that create conditions favorable for success of the community (e.g., anaerobiosis). Experiments that address these issues cannot be done in humans; they require an animal model that allows testing of the basic ecological principles that underpin the human microbiome. For over a decade, we have used the streptomycin-treated mouse model of intestinal colonization to characterize the functional role of Escherichia coli in the intestine. We learned that different E. coli strains execute different nutritional programs, allowing them to co-colonize the gut. Importantly, we learned that E. coli respires oxygen and thereby lowers the oxygen tension of the cecum, creating conditions that favor growth of the predominantly anaerobic microbial community. Therefore, we hypothesize that E. coli lives in symbiotic relationship with the anaerobes. Since anaerobes are sensitive to oxygen, we predict that the community composition of the intestine depends at least in part on the oxygen scavenging function contributed by E. coli. Since E. coli is unable to hydrolyze complex polysaccharides, which are the primary nutrient source in the gut, and because E. coli can grow only on the degradation products, we predict that anaerobic polysaccharide degradation releases simple sugars that cross-feed E. coli. And, since different E. coli strains execute different nutritional programs in the intestine, we predict that each E. coli strain will interact with distinct subpopulations of the microbial community. Here we outline a Research Plan designed to test these predictions. In Aim 1 we will use high-throughput sequencing of 16S rRNA gene tags and molecular phylogenetic analysis to examine the microbial composition of the streptomycin-treated mouse intestine as it is affected by E. coli colonization. Using isogenic E. coli strains that can and cannot respire oxygen, we will test the prediction that oxygen scavenging in the intestine affects the composition of the anaerobic microbial community. In addition, we will test the prediction that colonization of the intestine with different strains of E. coli, each of which consumes different nutrients in the intestine, will influence the microbial community composition. In Aim 2 we will test the prediction that different E. coli strains physically associate with different members of the microbial community by using 16S analysis of microbes sampled from the intestine by laser capture micro-dissection. Furthermore, we will characterize nutrient flow between E. coli and individual members of the intestinal microbial community in co-cultures. In Aim 3 we wil determine whether surface structures, including O-polysaccharides, flagella, and capsule, target E. coli to specific microhabitats where they could interact with different members of the microbiota. Thus we will characterize the interactions of E. coli with the microbial community in the intestine.